action of an epimerase which converts meso-DAP to LL-DAP ... meso-DAP, the DAP epimerase, was demon- ...... acid racemase, an enzyme causing intercon-.
JOURNAL OF
BACrERIOLOGY,lDeC.
Vol. 96, No. 6 Printed in U.S.A.
1968, p. 2099-2109 Copyright © 1968 American Society for Microbiology
Diaminopimelate Decarboxylase of Sporulating Bacteria DUANE P. GRANDGENETT1 AND D. P. STAHLY Department of Microbiology, University of Iowa, Iowa City, Iowa 52240
Received for publication 16 September 1968
The meso-diaminopimelate (DAP) decarboxylase of Bacillus licheniformis, a pyridoxal phosphate-requiring enzyme, was stabilized in vitro by 0.15 M sodium phosphate buffer (pH 7.0) containing 1 mm 2,3-dimercaptopropan-1-ol, 100 jig of pyridoxal phosphate per ml, and 3 mm DAP. When the meso-DAP concentration was varied, the enzyme in cell-free extracts of B. licheniformis exhibited MichaelisMenten kinetics. Pyridoxal phosphate was the only pyridoxine derivative which acted as a cofactor. The enzyme was subject to both inhibition and repression by L-lysine. The inhibitory effect of lysine was on the Km (meso-DAP). A maximum repression of about 20% was obtained. No significant inhibition or activation was produced by cadaverine, dipicolinic acid, phenylalanine, pyruvate, ethylenediaminetetraacetate, adenosine triphosphate, adenosine diphosphate, or adenosine monophosphate. When B. licheniformis was grown in an ammonium lactate-glucose-salts medium, an increase in DAP decarboxylase specific activity occurred during cellular growth with a maximal specific activity at the end of the exponential phase. As soon as growth ceased, the specific activity of the enzyme decreased to approximately one-half of the maximal specific activity and remained at this level thereafter. When B. cereus was grown in complex media, there was an increase in DAP decarboxylase specific activity up to the end of the exponential phase. Thereafter, the specific activity decreased to a nondetectable level in 4 hr. Dipicolinic acid synthesis was first detected 15 min later and was essentially complete after an additional 2.5 hr. The significance of the disappearance of DAP decarboxylase in B. cereus was discussed with regard to control of dipicolinic acid and spore mucopeptide biosynthesis.
The pathway for lysine and dipicolinic acid biosynthesis is illustrated in Fig. 1. The last enzyme in the lysine pathway, diaminopimelate decarboxylase, (EC 4.1.1.20; meso-2,6-diaminopimelate carboxy-lyase) is an enzyme that specifically decarboxylates the meso-isomer of diaminopimelate (DAP), yielding L-lysine. Previous work on the preparation and properties of this enzyme from Escherichia coli cells has been reviewed by Work (25) and White and Kelly (23). The regulation of lysine biosynthesis in nonsporulating bacteria is discussed in reviews by Datta and Gest (9) and Cohen (8). The DAP decarboxylase was shown to be repressed by Llysine in E. coli (16, 22), and Aerobacter aerogenes (22), but not in Bacillus megaterium or B. subtilis (22). White and Kelly (23) demonstrated Llysine inhibition of the enzyme in E. coli. Vinter (20) has demonstrated two periods of
maximal 14C-DAP incorporation into the spore mucopeptide of B. cereus. The first occurred before the formation of mildly refractive spores. This 14C-DAP was incorporated into the cells as both DAP and lysine. The second period of incorporation was denoted by an increase in the refractivity of the spores. This period of maximal 14C-DAP incorporation occurred during and after maximal dipicolinic acid synthesis. 14CDAP was not converted to 1"C-lysine during this period. Murrell and Warth (15) subsequently found a correlation between DAP content in the spore mucopeptide and heat resistance. DAP is found solely in the cortex mucopeptide layer of the spore. From Vinter's (20) finding, it appeared that the DAP decarboxylase may not be active at this second stage of 14C-DAP incorporation or that meso-DAP may be unavailable for decarboxylation. The latter situation could occur if the enzyme(s) responsible for incor1 Predoctoral Fellow (l-Fl-6M-37, 555-01) of the poration of meso-DAP into spore mucopeptide utilized all of the available intracellular mesoU.S. Public Health Service. 2099
GRANDGENE1T AND STAHLY
2100
J. BAcTERIOL.
-- L-Threonine 2ll 1 3 ( Pyruvate Dipicolinic acid - Dihydrodipicolinic acid 5 4 L- Isoleucine L-Methionine
L-Aspartate -Aspartyl phosphate-'-Aspartic/3semialdehyde ->Homoserine-1
Tetr.ahydrodipicolinic acid N-acetyl-e-keto-La-aminopimelate N-acetyl-L-a , diaminopimelate L-a,c -diaminopimelate 6 Meso-diaminopimelate --Spore mucopeptide
7 L-lysine
1) Aspartokinase; 2) Aspartic semialdehyde dehydrogenase; 3)Dihydrodipicolinic acid synthetase; 4) Dihydrodipicolinic reductase; 5) Dipicolinic acid synthetase; 6) Diaminopimelic acid epimerase 7) Diaminopimelic acid decarboxylase
FIG. 1. Pathway for biosynthesis of dipicolinic acid and lysine in sporulating bacteria
DAP. DAP already in the cell wall does not Klett-Summerson photoelectric colorimeter with a participate in the synthesis of the spore muco- no. 54 green filter (500 to 570 nm). The pH measurements were made with a Beckman Zeromatic pH peptide (20). meter. This study was conducted to characterize the Harvesting and breaking of organisms. Cells were DAP decarboxylase in cell-free extracts of sporu- harvested by a Sharples supercentrifuge at 4 C. The lating bacteria, to determine what type or types cells were suspended in a buffer containing 3 mm DAP of control are involved in the regulation of the (mixture of meso- and LL-DAP), 100lg of pyridoxal DAP decarboxylase during different stages of phosphate per ml, 1 mm 2,3-dimercaptopropan-l-ol development in Bacillus species, and to determine (BAL), and sodium phosphate buffer (0.15 M, pH 7.0). whether the nonconversion of '4C-DAP to 14C- The cells were broken in this buffer unless otherwise lysine during the time of dipicolinic acid syn- stated. The cell-free extracts were obtained by disruppassage through a French pressure thesis was due to a disappearance of DAP decar- tion of the cells by cell (American Instrument Co., Inc., Silver Spring, boxylase activity in B. cereus. Md.). The debris was removed by centrifugation at 20,000 X g for 20 min in a refrigerated International MATERIALS AND METHODS centrifuge model B-20. The cell-free extract thus obOrganisms used and culture conditionis. B. lichenii- tained was used for enzyme studies. Cell-free extracts formis strain A-5 and B. cereus (ATCC 10702) were were always prepared from late exponential-phase used as the test organisms throughout this study. The cells unless otherwise stated. Dipicolinic acid was measured by the method of defined medium and conditions employed in culturing B. licheniformis were those specified by Bernlohr and Janssen and associates (12). Protein concentrations were determined by the Novelli (5), with the modification that the stock glucose solution contained 10 g of CaCl2 2H20 per Biuret assay (11). Meso-DAP separationi from a racemic mixture. liter. The complex medium and conditions employed in culturing B. cereus were described by Vinter (20). Meso-DAP was isolated from a mixture of meso- and Unless otherwise specified, these were the media LL-isomers by fractional crystallization from aqueous ethyl alcohol as described by Work (25). Ethyl alcohol employed. For repression studies with B. cereus, the orga- was added to an aqueous solution (25 mg/ml) of a nism was grown on Lysine Assay Medium (Difco). mixture of meso- and LL-isomers until there was slight The growth conditions were the same for the organism permanent turbidity at room temperature. After as in the other medium employed (20). The Lysine standing for 30 min, the precipitate was collected, Assay Medium was used as indicated by the manu- dissolved in water, and reprecipitated with ethyl alcohol. Several more precipitations resulted in profacturer except that it was diluted by a factor of 10. Cultures were routinely examined by observing wet duction of the meso-isomer containing none of the LL-isomer as determined by descending paper mounts with a Zeiss phase-contrast microscope. Turbidity of cultures was determined by using a chromatography (1).
VOL. 96, 1968
Chromatography. Samples
DIAMINOPIMELIC ACID METABOLISM were
applied
on
What-
and subjected to descending chromatography. The spots were developed with ninhydrin in acetone (0.1%, w/v). The solvent was methanolwater-10 N HCl-pyridine [80:17.5:2.5: 10, v/v (1)]. Measurement of enzymatic activity. DAP decarboxylase activity was usually determined by measuring the rate of disappearance of meso-DAP by using a specific colorimetric method in which lysine does not interfere (24). Unless otherwise stated, each reaction tube contained the following compounds: BAL, (1 mM); pyridoxal phosphate, 30 ,ug/ml; 0.2 M sodium phosphate buffer, pH 7.3; meso-DAP (38.9 mM); enzyme solution (0.1 to 0.5 ml); and water in a final volume of 1 ml. The enzyme solution was added after the other components had been mixed and equilibrated at 37 C. The tubes were shaken periodically and samples were removed at intervals to tubes containing acetic acid (4 ml) and water (0.4 ml). When all the samples had been taken, 0.5 ml of ninhydrin reagent (250 mg of ninhydrin dissolved in 4 ml of 0.6 M phosphoric acid and 6 ml of acetic acid) was added to each tube and the mixture was heated at 37 C for 90 min. The absorbancy of the mixtures was then measured at 440 nm on a Zeiss spectrophotometer (PMQII). Enzymatic activity was also measured manometrically in a Warburg apparatus at 37 C in an atmosphere of air. There was no difference in the rate of decarboxylation when the assay was performed in an atmosphere of nitrogen rather than air. The same concentrations of reactants were used as above, except that the final volume was increased 2.5-fold. As indicated in the experimental section, occasionally phosphate buffer, pH 6.8, was substituted for the pH 7.3 buffer. Since an accurate correction for retention of CO2 cannot be made at these pH values (19), the colorimetric assay was used rather than the manometric assay unless otherwise stated. The rate of disappearance of meso-DAP was correlated with the rate of evolution of CO2 by assuming that decarboxylation of 1 mole of meso-DAP gave 22.4 liters of CO2. All reaction rates were calculated from initial velocities. When the enzyme was assayed at pH 7.3 by both the colorimetric method and the manometric method, the activities were the same provided that the pH in the Warburg flask was lowered to pH 5.0 after completion of the reaction to evolve the total CO2 formed. Materials. The following materials were obtained from the Sigma Chemical Co. (St. Louis, Mo.): a-e-DAP (mixture of meso- and LL-DAP), BAL, DLpenicillamine, cadaverine, L-lysine, L-phenylalanine, pyruvic acid (potassium salt type III), pyridoxal phosphate, pyridoxine monohydrochloride, pyridoxamine- 2HCI, pyridoxamine-5'-phosphate HCI, pyridoxal HCI, and lysine decarboxylase (type I powder from Bacterium cadaveris). Dipicolinic acid was obtained from Aldrich Chemical Co., Inc., Milwaukee, Wis., and peptone and Lysine Assay Medium were from Difco. C. Gilvarg kindly provided meso- and LL-DAP for use as chromatographic standards. man no.
1
paper
2101
RESULTS Requirement for enzymatic activity. DAP decarboxylase activity in cell-free extracts of B. licheniformis showed a marked dependence upon pyridoxal phosphate and meso-DAP and was definitely stimulated by BAL (Table 1). There was a direct relationship between the rate of disappearance of meso-DAP and the concentration of the enzyme. At all concentrations of enzyme tested, the disappearance of mesoDAP was linear up to 40 min at the saturating concentration of substrate (38.9 mM). During manometric assay, a gas was evolved, the volume of which was directly related to the enzyme concentration. The gas evolved was absorbed by KOH and, therefore, was assumed to be carbon dioxide. Meso-DAP conversion to Llysine was demonstrated by paper chromatography (Fig. 2). To verify further that the product was lysine, lysine decarboxylase was added to the reaction tube which then converted the Llysine to cadaverine. Apparently, there was no active lysine decarboxylase in the cell-free extracts under these conditions as demonstrated by the lack of L-lysine conversion to cadaverine in the absence of added lysine decarboxylase. In addition to the DAP decarboxylase, the action of an epimerase which converts meso-DAP to LL-DAP was apparent. Effect of meso-DAP concentration on enzymatic activity. When varying the substrate concentration, the DAP decarboxylase of B. licheniformis demonstrated Michaelis-Menten kinetics with a Km (meso-DAP) of 6 to 10 mm. Some variation occurred between experiments. This high Km may have been due to the presence of another enzyme(s) in the cell-free extract which binds meso-DAP. One other enzyme which binds meso-DAP, the DAP epimerase, was demonTABLE 1. Requirements for DAP decarboxylase activity in cell-free extracts ofB. licheniformis Component
Specific activ-
ityG
0.032 Complete6 ..................... Minus pyridoxal phosphate ............... 0.009 0.025 Minus BAL ..................... Minus cell-free extract .................... 0.000 Plus boiled cell-free extractc .............. 0.000 a Expressed as micromoles of DAP decomposed per minute per milligram of protein. b Complete reaction mixture described in the Materials and Methods. c Boiled cell-free extract was added in place of cell-free extract in the reaction mixture.
2102
GRANDGENETI AND STAHLY
strated to be present by paper chromatography
(Fig. 2).
Influence of pH on enzyme activity. DAP decarboxylase in cell-free extracts from B. licheniformis cells exhibited optimal activity at pH 7.30. Stability studies of DAP decarboxylase in cellfree extracts of B. licheniformis. White and Kelly (23) reported that DAP decarboxylase in cell-free extracts of E. coli lost activity more quickly at 2 C when pyridoxal phosphate was added than when the cofactor was absent. Studies were conducted to determine what effect pyridoxal phosphate had on stability of the DAP decarboxylase of B. licheniformis. The effect of the substrate, DAP, and the product, lysine, on storage stability was also determined (Table 2). When DAP (mixture of meso- and LL-DAP; 3 mm) and pyridoxal phosphate (100 ,g/ml) were present at breakage, 96% of the original activity was retained after 4 days. These concentrations were the minimal amounts required for stabilization. The effect of pyridoxal phosphate, DAP, and lysine on
J. BAcrERIOL.
storage stability of cell-free extract from cells broken in their absence was also determined (Table 2). Neither DAP nor lysine had any stabilizing effect by themselves; pyridoxal phosphate by itself provided some stability. No inactivation occurred in the absence of pyridoxal phosphate and DAP during the breakage process per se, but addition of these substances after breakage did not provide the same degree of enzyme stability as they did when they were also present during the time of cell breakage. A possible explanation for this discrepancy is that, during breakage of the cells in the absence of pyridoxal phosphate and DAP, some of the enzyme molecules begin to unfold. If assayed immediately after breakage, they prove to be active; but, even though the stabilizers (DAP and pyridoxal phosphate) are then added, some of these molecules continue to be altered to the point at which they are no longer active. When BAL was absent at breakage, even with pyridoxal phosphate and DAP present, the enzymatic activity decreased 35% after 1 day of storage at -20 C. No activity was observed
Chromotogram of Reaction Mixture
Extract + meso-DAP Extract L- lysine Extract
meso-DAP
decarboxylase
meso-DAP
LL-DAP
L-lvsine
Cadaverine
1I 0O0 0O0 0
0
6 0
1\'
0 Solvent:
0
methanol-water-ION HCl-pyridine (80:17.5:2.5:10, by volume).
Rmeso-DAP neso-diaminopimelic acid (DAP) - 1.0 - 1.23 LL-diaminopimelic acid - 1.87 L-lysine cadaverine
-
2.50
2. Chromatogram of reaction mixture with the cell-free extract of B. licheniformis. After 30 min of incubation, S guiters of each reaction mixture was applied to a chromatogram and subjected to descending chromatography.
rrFIG.
VOL. 96, 1968
DIAMINOPIMELIC ACID METABOLISM
2103
TABLE 2. Stability studies on the cell-free extracts of B. licheniformisa Activity (% of original activity) Stability studies of cell-free extract
Broken in the presence of 3 mm DAP + 100 ,ug/ml of PLPc (undialyzed) ............. Broken in the presence of 3 mm DAP + 100 Ag PLP and dialyzed against the same medium .......................... Crude extract from cells broken in buffer only Without dialysis .......................... Dialysis with buffer only .................. Without dialysis + 100 ,Ag of PLP/ml added. Dialyzed against 100 ,Ag of PLP/ml ........ Dialyzed against 3 mm DAP ............... Dialyzed against 3 mm DAP + 100 Mg of
PLP/ml ................................. Dialyzed against 3 mM L-lysine ............ Dialyzed against 3 mM L-lysine + 100 jg of PLP/ml ................................. Dialyzed against 3 mm DAP + 3 mM Llysine + 100 jug of PLP/ml ..............
0 daysb
1 day
2 days
3 days
4 days
100
100
98
95
96
100
100
97
95
94
100 23 100 87 18
64 0 80 51 0
59 0 80 45 0
51 0 75
45
94 24
81 0
78 0
79 0
92
81
71
69
85
82
77
73
70
0
a All extracts contained 0.15 M sodium phosphate buffer at pH 7.0 and 1 mm 2, 3-dimercaptopropanol. Dialysis was performed against the same medium for 7 hr at 4 C. Extracts were stored at -20 C. I Time of storage of cell-free extract. c Pryridoxal phosphate.
on the 2nd day. A concentration of BAL greater and its derivatives, pyridoxal, pyridoxamine, than 1 mm provided no additional stabilizing pyridoxal-5'-phosphate, and pyridoxamine-5'phosphate, all at a concentration of 30 Ag/ml, effect. In the presence of DAP, pyridoxal phosphate, were tested as cofactors for the DAP decarboxyland BAL, the degree of stability was determined ase of B. licheniformis. The cells were broken as at various pH values. The pH optimum for described in Materials and Methods, but no stability was between 6.7 and 7.3. The molarity pyridoxal phosphate was present in the susof the sodium phosphate buffer for the best pending medium. None of the pyridoxine destabilization was 0.15 to 0.20 M. The enzymatic rivatives were active as cofactors, except pyridoxal activity was stable for at least 1 month in whole phosphate. Under these conditions, the concentration of added pyridoxal phosphate required cells at -20 C. Reactivation studies of DAP decarboxylase. to achieve Vmax varied from 1 to 2.0 ,ug/ml As indicated in Table 2, cell-free extract pre- with the different cell-free extracts. Effect of various compounds on B. licheniformis pared from B. licheniformis cells suspended and dialyzed in sodium phosphate buffer with 1 DAP decarboxylase activity. Pyridoxine, pyrimM BAL lost enzymatic activity rapidly. Differ- doxal, pyridoxamine, and pyridoxamine- 5'ent methods were used in an attempt to reacti- phosphate, each at 30 ,ug/ml, did not inhibit vate the partially inactivated enzyme. Optimal the enzymatic activity when tested in the presreactivation of the inactivated enzyme was ob- ence of various concentrations of pyridoxal tained by placing the dialyzed extract in the pres- phosphate (1, 10, and 30 ,ug/ml). Inhibition ence of 300 j,g of pyridoxal phosphate per ml of activity was caused by DL-penicillamine and 6 mm DAP (mixture of meso- and LL-DAP). which was prevented by increased concentrations The greatest amount of reactivation was 30% of pyridoxal phosphate (Table 3). DL-penicillawhen stored at -20 C for 24 hr, either in the mine has been reported to inhibit decarboxylafrozen state or as a liquid in glycerol (50:50, tion of amino acids through the formation of v/v). Storage at 4 C or higher was impractical thiozolidinecarboxylic acid derivative with pyribecause the enzyme is inactivated at these tem- doxal phosphate (14). L-Lysine inhibits the DAP decarboxylase in peratures. Effect of various derivatives of pyridoxine as B. licheniformis (Fig. 3). At very high lysine cofactors for DAP decarboxylase. Pyridoxine concentration (80 mM), inhibition was complete.
2104
GRANDGENE1T AND STAHLY
TABLE 3. Effect of DL-penicillamine on enzymatic activitya Pyridoxal phosphate concn (mM)
Penicillamine concn (mm)
Activity (% of control)
0 6 6 6 6 3 0
100 90.5 68.5 38.0 00.0 00.0 30.0
8 (control) 8 4 2 0 0 0
Reaction mixture described in Materials and Methods. Only the concentrations of pyridoxal phosphate and penicillamine were changed. All of the above concentrations of pyridoxal phosphate were sufficient to achieve maximal activity when no penicillamine was present. The specific activity of the control was equal to 0.036,umole of DAP decomposed per min per mg of protein.
Variation of the pH between 6.75 and 7.85 in the reaction mixture had no effect on the degree of inhibition of the enzymatic activity by lysine. Manometric studies also verified lysine inhibition. When 10 mm lysine was present in the Warburg flask in the presence of 38.9 mM meso-DAP, it caused a 21 % decrease in the amount of CO2 evolved. Other compounds. Several other compounds were tested for their ability to inhibit or activate the enzyme: BAL (2, 4, and 6 mM); dipicolinic acid (10 mM), pyruvate (20 mM); phenylalanine (20 mM); cadaverine (20 mM); adenosine triphosphate (10 mM); adenosine diphosphate (10 mM); adenosine monophosphate (10 mM); and ethylenediaminetetraacetate (EDTA, 1 and 5 mM). The control reaction mixture contained 30 ,ug of pyridoxal phosphate per ml with 1 mm BAL. The substrate concentrations used were at saturating (38.9 mM) and unsaturating (9.0 mM) levels. None of the compounds caused inhibition, and EDTA was the only compound tested which produced increased activity (5 to 6%). Repression of DAP decarboxylase by L-lysine in B. licheniformis. Lysine was added to cells growing exponentially in glucose-ammonium lactate-salts-medium (5), and samples were taken immediately and at periodic intervals thereafter from both the control flask and the flask containing lysine. The cells were washed three times with 0.05 M sodium phosphate buffer (pH 7.0) and broken, and the extracts were tested for enzymatic activity. DAP decarboxylase activity from cells which were grown in the presence of 10 mM lysine had a maximum specific activity which was about 20% lower than that from cells grown in the absence of lysine (Fig. 5). Lysine at 20 mm concentration caused no greater 0.32 - I 6mM Lysine 0.28 V 'pmoles DAP decomposed/30min. [S],upmoles DAP/mi 0.24
20
[L- lysine](mM) FIG. 3. L-Lysine inhibition of DAP decarboxylase from B. licheniformis. The reaction mixture was the same as described in Materials and Methods, except for varying the substrate (mesoDAP) and the inhibitor (L-lysine) as indicated. The specific activity of the control (without lysine present) was 0.037 jsmole of DAP decomposed per min per mg ofprotein.
J. BACTERIOL.
0.20
F
0.16 0.12
/~~
0.08
-10
0
0o /
I
Ke 18.0mM No I
~~---K",:9.1mb1
W
I-, '0o
-
7>z-* 1.0
2.0
IIS]
When the substrate concentration was varied at a constant lysine concentration (6 mM), the Vmax was essentially unchanged, but the Km (meso-DAP) was increased (Fig. 4). Thus it appears that lysine is a competitive inhibitor.
FIG. 4. Lineweaver-Burk plot of L-lysine inhibition of DAP decarboxylase from B. licheniformis. The reaction mixture was the same as described in Materials and Methods, except that the substrate concentration was varied as indicated. The reaction mixture contained 8.6 mg of protein/ml.
'VOL. 96, 1968
DIAMINOPIMELIC ACID METABOLISM
2105
DAP decarboxylase activity as correlated with the stage of development in B. licheniformis. The DAP decarboxylase activity, as related to stage of development in B. lichenijbrmis, is shown in Fig. 6. There was an increase in specific activity during growth, with its peak occurring at the end of the exponential phase at about 5 If 200 / Uk 1mM L-Iysine A% 03 hr. Soon thereafter, the specific activity of the enzyme decreased to approximately one-half of the maximal activity and remained at this level thereafter. After 10 hr, refractile endospores rowth 0.02 0 ..0~~~~~~~~0 began to appear, but 5 hr later only 50 to 60%. of the cells formed refractile endospores. Lysis of nonsporulated cells was also apparent. The i _ ~~~~~~~~~~~~0.01 slight increase in turbidity, always observed between 6 and 10 hr, is not due to an increase in cell numbers but can be accounted for by intracellular changes in the presporulating cells. 0 1 2 3 4 5 6 7 8 When B. licheniformis was grown in the usual Age of Culture of Bacillus licheniformis (hr) weakly buffered ammonium lactate-glucoseFIG. 5. Repression of DAP decarboxylase in B. salts medium, a decrease in pH occurred, followed licheniformis growing in glucose-ammonium lactate- by an increase (Fig. 6). To rule out the possisalts medium. Arrow indicates time of addition of L- bility that a change in pH in the growth medium lysine. Control curve indicates specific activity of cells affects the enzymatic activity, an experiment grown in the absence of L-lysine. Growth and sporula- was conducted identical to that above, except tion patterns were the same with or without lysine that the growth medium was maintained at pH present. 7.0 i1: 0.2 with 0.2 M sodium phosphate buffer. There were no differences in enzymatic activity decrease in activity. This experiment was repeated between the cell-free extracts from cells grown four times. in strongly and weakly buffered media. The Several control experiments were performed growth and sporulation patterns in both media to rule out the possibility that lysine might be were the same. present in the cell-free extracts. It was found In B. licheniformis, sporulation is asynchronous that the activities of the cell-free extracts from and incomplete. It was apparent that DAP the cells grown in the presence and absence of decarboxylase was definitely present during lysine were the same after dialysis as before dialysis. Also, cells harvested immediately after addition of lysine (before repression could be expressed) had the same enzymatic activity as those grown in the absence of lysine. Both of the above controls demonstrated that lysine apparently was not present in the extracts to cause inhibition of the DAP decarboxylase and, therefore, the decrease in activity was probably due to
repression. When the initial culture inoculum was grown in the presence of 10 mm lysine and 10 mM lysine was present also in the growth flask, the specific activity was 25% lower than that of the enzyme from cells grown in the control flask which did not contain lysine. The presence of lysine in the growth flasks neither increased nor decreased the rate of cellular growth. Therefore, the rate of synthesis of lysine in the absence of lysine was not growthlimiting. Also, excess lysine apparently does not cause a growth-limiting deficiency of any other metabolite required for growth.
O
2
4
6
8
10 12 14
16
Age of Culture of Bacillus licheniformis(hr) FIG. 6. DAP decarboxylase activity of B. licheniformis at various stages ofdevelopment. Growth medium employed was the normal weakly buffered glucoseammonium lactate-salts medium described in Materials and Methods. Samples were taken at the indicated times and the specific activity was determined.
2106
GRANDGENETT AND STAHLY
sporulation, but, since many of the cells were not sporulating, it could not be ruled out that the DAP decarboxylase was absent from the sporulating cells. To explain Vinter's finding (20) of nonconversion of DAP to lysine at a late stage in sporulation, it is necessary to have an organism which sporulates nearly 100% with a high degree of synchrony. Therefore, the above experiments were repeated with B. cereus (ATCC 10702), which possesses these characteristics. This strain of B. cereus is identical with that used by Vinter (20), and thus results concerning DAP decarboxylase activity can be directly correlated with Vinter's in vivo results concerning conversion of DAP to lysine. DAP decarboxylase activity correlated with the stages of development in B. cereus. The specific activity of DAP decarboxylase, as related to the stage of development of B. cereus, is shown in Fig. 7. There was an increase in specific activity as growth continued, with the maximal specific activity occurring at the end of the exponential phase. Thereafter, the specific activity decreased to a nondetectable level in 4 hr. Dipicolinic acid synthesis was first detected 15 min later and was essentially complete 2.5 hr thereafter. Sporulation was much more synchronized and complete with B. cereus than with B. licheniformis. This synchrony was evidenced by the rapid increase in dipicolinic acid, by nearly all of the cells becoming completely refractile within 30 min as obser-ved by phase-contrast microscopy, and by apparent lack of cell lysis. Disappearance of enzymatic activity in the late-sporulating cell-free extracts could have been due to an inhibitor. When cell-free extracts prepared from 9-hr cells, which possessed no detectable enzyme activity, were added to cellfree extracts from 3-hr cells in the reaction mixture, there was no observable decrease in activity. Thus, no enzyme inhibitor has yet been detectable in sporulating cell extracts. Properties of DAP decarboxylase of B. cereus. The properties of the DAP decarboxylase of B. cereus have not as yet been studied as extensively as those of B. licheniformis. The following properties of the enzyme were observed. The pH optimum for enzymatic activity was 7.05; the enzyme required BAL for full activity; and the activity was proportional to the enzyme concentration. The enzyme was stable for at least 1 day in cell-free extracts when conditions for breakage of B. licheniformis cells were employed. Dialysis of this cell-free extract for 7 hr against 0.15 M sodium phosphate buffer (pH 7.0), containing 3 mm DAP, 1 mm BAL,
J. BAcrERIOL.
Nl-
44
t_-
"I,,
k7tp as 1-ft
Z, Zt.
CZi"I Itbt tt
0 2 4 6 8 10 Age of Culture of Bacillus cereus (hr) FIG. 7. DAP decarboxylase activity in B. cereus at various stages of development. The complex growth medium used was that described in Materials and Methods. At the indicated times, the specific activity and dipicolinic acid content were determined.
and 100 ,ug of pyridoxal phosphate per ml, resulted in a 30% decrease in activity. Results from preliminary experiments indicate that DAP decarboxylase of B. cereus is subject to both inhibition and repression by L-lysine. Lysine at 10 mm concentration caused a 15% decrease in activity when the meso-DAP concentration was 38.9 mm. When the cells were grown in the complex medium (20) which contains lysine, the maximal specific activity obtained was 0.016 ,umoles of meso-DAP decomposed per min per mg of protein. However, when growth occurred in modified Lysine Assay Medium, a rich medium lacking lysine, the maximal specific activity was increased approximately fourfold. When 20 mm lysine was added to this medium, the maximal specific activity was decreased about fourfold. Lysine had no effect on the growth rate and on the time of appearance of refractile spores. DISCUsSION
The pathway for dipicolinic acid biosynthesis (Fig. 1) was elucidated by Bach and Gilvarg (3) and Fukuda and Gilvarg (10). The synthesis of dipicolonic acid in sporulating bacteria occurs as a branch of the lysine pathway (Fig. 1). Therefore, the regulation of the synthesis of dipicolinic acid and lysine might share certain characteristics with the regulatory patterns of a number of other branched pathways which are controlled by end products (8, 9). In these path-
VOL. 96, 1968
DIAMINOPIMELIC ACID METABOLISM
ways in which several end-product metabolites are derived from a common precursor, operation of the two regulatory mechanisms, repression and end-product inhibition, poses a problem. Over-production of one end-product metabolite might lead to a reduction in the rate of production of the common intermediate to a level below that needed for the optimal biosynthesis of another end-product metabolite. This problem could arise in Bacillus species by the fact that dipicolinic acid is synthesized from dihydrodipicolinic acid, which is also an intermediate in the synthesis of lysine. No problem occurs, though, since a continued flow of carbon is available for dipicolinic acid synthesis during sporulation (2). The aspartokinase, aspartic semialdehyde dehydrogenase, and dihydrodipicolinic acid synthetase are all apparently functioning at the time of dipicolinic acid syn-
2107
ance of the DAP decarboxylase could have been due to asynchronous and incomplete sporulation. Although no data were presented, White et al. (22) reported that, in B. subtilis, DAP decarboxylase specific activity decreased rapidly after reaching a maximum at the end of the growth phase. No information was given as to the extent of this decrease in activity. In summary, nonconversion of meso-DAP to lysine in vivo at a late stage of sporulation can be accounted for by disappearance of DAP decarboxylase activity in cell-free extracts, at least in B. cereus. This lack of DAP decarboxylase activity during sporulation could also account for the observation of Aronson and others (2) that very little of the radioactivity from 14Cf aspartate was found in lysine at this stage. The fact that lysine biosynthesis is blocked during spore maturation by a disappearance in thesis. DAP decarboxylase activity does not rule out Dihydrodipicolinic acid synthetase, the first the possibility that another block may occur enzyme in the lysine-dipicolinic acid pathway, earlier in the pathway. The disappearance of is not inhibited or repressed in spore-formers DAP decarboxylase could be an indirect cause by lysine, DAP, or dipicolinic acid (2, 7; D. P. of this proposed block by allowing the accumulaStahly, Bacteriol. Proc., p. 141, 1968). This tion of DAP, which might inhibit or repress (or differs from the regulation in E. coli which pos- both) dihydrodipicolinic acid reductase. This sesses a dihydrodipicolinic acid synthetase which type of control would insure that an adequate is inhibited by lysine (26). supply of dihydrodipicolinic acid was available What happens to the flow of carbon from for dipicolinic acid synthesis, while avoiding dihydrodipicolinic acid to lysine at the time of the unnecessary synthesis of the metabolic indipicolinic acid synthesis? Aronson (2) has termediates between dihydrodipicolinic acid demonstrated in B. cereus that, when 14C- and DAP. In order for this type of control to aspartic acid was added to sporulating cells at be exerted on dihydrodipicolinic acid reductase, the time of dipicolinic acid synthesis, very little the end product(s) would have to accumulate in of the radioactivity was converted to lysine as vivo. The proposed accumulation of DAP during compared to the conversion to other amino acids synthesized from aspartate or to the con- sporulation, in addition to perhaps playing a version to lysine occurring in young cells. Vinter role in regulation of dipicolinate biosynthesis, (20) earlier found that 14C-DAP was not con- may also allow DAP to be more readily taken verted to 14C-lysine during dipicolinic acid up into the cortex mucopeptide layer of the spore synthesis in B. cereus. As indicated previously, (20, 21). both of these observations could be accounted The disappearance of the DAP decarboxylase for if DAP decarboxylase activity disappears activity in B. cereus could be due to a variety of or if all of the available DAP is incorporated factors. Among the possible causes of enzyme into the spore mucopeptide at this time. In disappearance are low- and high-molecularB. cereus, we found that the DAP decarboxylase weight inhibitors, cofactor deficiency, and specific activity did decrease to a nondetectable proteolysis of the enzyme (alone or coupled with level, the disappearance slightly preceding dipi- repression of enzyme synthesis). These possible colinic acid synthesis. Thus, the apparent block causes may occur by themselves, or several may in lysine biosynthesis in vivo (2, 20) during act together to account for the disappearance of sporulation can be accounted for by the observed the enzyme activity. disappearance of DAP decarboxylase activity. Dialysis of the inactive extracts from sporulatThe DAP decarboxylase in cell-free extracts ing cells resulted in no increase in activity, thus from sporulating cells of B. licheniformis (this eliminating the possibility that a low-molecularstudy) and B. sphaericus (17) had approximately weight inhibitor was responsible for the enzyme one-half of the highest specific activity achieved inactivity. The possibility of a strongly bound in the vegetative cells. But the lack of disappear- inhibitor cannot be ruled out. Addition of inactive
'2108
GRANDGENETT AND STAHLY
cell-free extract from sporulating cells to active cell-free extract from late-exponential-phase cells in reaction mixture resulted in no observable decrease in activity. Thus, it seems unlikely that a macromolecular enzyme inhibitor was present in the sporulating cell extract. However, the possible presence of an inhibitor which is inactivated upon cell breakage, or which is stable only when bound to its specific enzyme, cannot be ruled out. A proteinaceous inhibitor of ornithine transcarbamylase that may be of this type was found in Saccharomyces cerevisiae by Bechet and Wiame (4). The concentration of pyridoxal phosphate in vivo may have some function in the disappearance of DAP decarboxylase activity during sporulation. For example, if a pyridoxal phosphate deficiency developed in vivo during sporulation, and if the cofactor was required for enzyme stability, then the observed decrease in enzymatic activity presumably could be due simply to enzyme inactivation. It has been shown in this study that pyridoxal phosphate is required for in vitro stability of the DAP decarboxylase in B. licheniformis. Complete or partial inactivation of the DAP decarboxylase by proteolysis in B. cereus may be responsible for the observed decrease in enzymatic activity. In B. cereus (13) and B. licheniformis (6), a protease is induced after cessation of growth. Spudich and Kornberg (J. Biol. Chem., in press) has demonstrated, by use of 3H-phenylalanine incorporation methods, that there is a 20% protein turnover per hour during sporulation in B. subtilis. Evidence that proteases are involved in modification of vegetative enzymes was provided by Sadoff (Bacteriol. Proc., p. 25, 1968). Deutscher and Kornberg (J. Biol. Chem., in press) presented evidence that the disappearance of inosine monophosphate dehydrogenase in sporulating extracts of B. subtilis may be due to proteolytic digestion of the enzyme. Therefore, it is conceivable that a protease modifies the DAP decarboxylase in B. cereus, changing it to an inactive form. Disappearance of enzyme activity during the time after growth cessation and appearance of spores, if caused by proteolysis alone without curtailing enzyme synthesis, would be energetically very uneconomical for the cell. Repression of the DAP decarboxylase followed by proteolysis would be less costly. The observed decrease in DAP decarboxylase activity, which took 4 hr, could be accounted for if complete or nearly complete repression occurred in B. cereus, and if there were a 25 %,o turnover of protein per hr. Results from preliminary experiments with B. cereus demonstrated that a fourfold decrease
J. BACTERIOL.
in maximal specific activity of the DAP decarboxylase occurred when cells were grown in the presence of lysine. In B. licheniformis, lysine was found to repress the enzyme about 20%. To our knowledge, this is the first report of lysine repression of the DAP decarboxylase in Bacillus species. Inhibition of the B. licheniformis DAP decarboxylase was affected by L-lysine. The only other report of lysine inhibition of this enzyme was in E. coli (23). Until information is obtained concerning the lysine and the DAP intracellular pool size in B. licheniformis, no conclusions can be made concerning lysine control of DAP decarboxylase in vivo. It is possible that lysine control of DAP decarboxylase by inhibition and repression may serve a valuable function in vivo. As shown in Fig. 1, meso-DAP is not only the precurser of lysine, but is also one of the components of spore mucopeptide. If lysine was available to the cell in excess, then by inhibition and repression of the DAP decarboxylase, lysine biosynthesis would be retarded. Also, DAP would then be more available for mucopeptide synthesis. ACKNOWLEDGMENT This work was supported by grant GB-5898 from the National Science Foundation. 1.
2.
3.
4.
5.
6. 7.
8.
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in microorganisms, p. 105-126. In C. E. Clifton, S. Faffel, and M. P. Stan (ed.), Annual review of microbiology, vol. 19. Annual Reviews, Inc. Palo Alto, Calif. Datta, P., and H. Gest. 1965. Alternative patterns of end product control in biosynthesis of amino acids of the aspartic family. Nature 203:12591261. Fukuda, A., and C. Gilvarg. 1968. The relationship of dipicolinate and lysine biosynthesis in Bacillus megaterium. J. Biol. Chem. 243:38713878. Gornall, A. G., C. J. Bardawill, and M. D. Maxima. 1949. Determination of serum protein by means of the biuret reaction. J. Biol. Chem. 117:751-766. Janssen, F. W., A. J. Lund, and L. E. Anderson. 1958. Colorimetric assay of dipicolinic acid in bacterial spores. Science 127:26-31. Levisohn, L., and A. I. Aronson. 1967. Regulation of extracellular protease production in Bacillus cereus. J. Bacteriol. 93:1023-1030. Mardaskev, V. 1963. Some aspects of amino acid decarboxylase inhibition, p. 277-290. In Snell, E. E., P. M. Fasella, and A. R. Fanelli (ed.), Chemical and biological aspects of pyridoxal catalysis. The Macmillan Co., New York. Murrell, W. G., and A. D. Warth. 1965. Composition and heat resistance of bacterial spores, p. 1-24. In L. L. Campbell and H. 0. Halvorson (ed.), Spores III. American Society for Microbiology, Ann Arbor, Mich. Patte, J. C., T. Loviny, and G. N. Cohen. 1962. Repression de la decarboxylase de la acide meso-a, e-diaminopimelique par la L-lysine, Escherichia coli. Biochim. Biophys. Acta 58:359-360. Powell, J. F., and R. E. Strange. 1957. a,e-
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diaminopimelic acid metabolism and sporulation in Bacillus sphaericus. Biochem. J. 65:700708. Sundharadas, G., and C. Gilvarg. 1967. Biosynthesis of a,e-diaminopimelic acid in Bacillus megaterium. J. Biol. Chem. 242:3983-3984. Umbreit, W. W., R. H. Burris, and J. D. Stauffer. 1959. Manometric techniques, p. 30. Burgess Publishing Co., Minneapolis. Vinter, V., 1963a. Spores of microorganisms XII. Non-participation of the pre-existing sporangial cell wall in the formation of spore envelopes and the gradual synthesis of DAPcontaining structures during sporogenesis of bacilli. Folia Microbiol. 8:147-155. Vinter, V. 1963b. Spores of microorganisms. Chloroamphenicol-sensitive and penicillin-resistant incorporation of C'4-diaminopimelic acid in sporulating cells of Bacillus cereus. Experientia 19:307-308. White, P. J., B. Kelley, A. Suffling, and E. Work. 1964. Variation of activity of bacterial diaminopimelic acid decarboxylase under different conditions of growth. Biochem. J. 91:600-610. White, P. J., and B. Kelly. 1965. Purification and properties of diaminopimelic acid decarboxylase from Escherichia coli. Biochem. J. 96:75-84. Work, E. 1957. Reaction of ninhydrin in acid solution with straight-chain amino acids containing two amino groups and its application to the estimation of a, E-diaminopimelic acid. Biochem. J. 67:416-423. Work, E. 1962. Diaminopimelic decarboxylase, p. 864-870. In S. P. Colowick and N. 0. Kaplan (ed.), Methods of enzymology, vol. 5. Academic Press, Inc., New York. Yugari, Y., and C. Gilvarg. 1965. The condensation step in diaminopimelic acid synthesis. J. Biol. Chem. 240:4710-4716.
